The 300kDa insulin-like growth factor II/ cation-independent mannose 6-phosphate receptor (IGF-II/CI-MPR) and the 46kDa cation-dependent MPR (CD-MPR) interact with a number of different proteins by virtue of their ability to bind mannose 6-phosphate (Man-6-P) residues with high (nM) affinity. The MPRs play a key role in delivering newly synthesized lysosomal enzymes to the lysosome in all higher eukaryotic cells and disruption of this targeting pathway results in the most severe of the human lysosomal storage disorders, mucolipidosis II. In addition to lysosomal enzymes, several growth factors contain Man-6-P and their interaction with the MPRs can result in the growth factor's activation or degradation. The multifunctional IGF-II/CI-MPR also binds several non-Man-6-P-containing ligands (IGF-II and retinoic acid) with high affinity. The observation that the IGF-II/CI-MPR regulates normal fetal growth by modulating IGF-II levels and undergoes allelic loss and mutation in a variety of human cancers implicates the IGF-II/CI-MPR as a tumor suppressor gene. Furthermore, the binding of one ligand to the IGF-II/CI-MPR can influence the binding of a second ligand: lysosomal enzymes impair IGF-II binding and IGF-II binding can inhibit lysosomal enzyme binding. Despite the ubiquitous expression of these receptors and the number of different molecules with which they interact, little is known about the molecular basis by which the MPRs recognize their diverse ligands. The five specific aims of this proposal will determine the three-dimensional structure of the Man-6-P binding sites of the IGF-II/CI-MPR and CD-MPR under various conditions which are physiologically relevant (aims 1 & 3), which residues of the Man-6-P binding site of the CD-MPR and IGF-II/CI-MPR are essential for phosphomonoester and phosphodiester recognition (aims 2 & 4), and the three-dimensional structure of the full-length, membrane-associated CD-MPR (aim 5). To achieve these objectives, soluble fragments encompassing the Man-6-P binding sites of both MPRs and the full-length CD-MPR will be produced in milligram amounts in a baculovirus expression system and their three-dimensional structure determined by X-ray crystallography. Mutagenesis studies coupled with binding affinity determinations will be conducted to verify the role of specific residues in carbohydrate recognition and to determine the effect of ligand occupancy on IGF-II/CI-MPR activity. These studies will address our long-range goal, which is to understand at the structural level the mechanism by which these essential receptors carry out their diverse biological functions.